A polypeptide targeting HPV16 E6 protein and its preparation and application

Abstract

The application discloses a polypeptide targeting HPV16 E6 protein and a preparation method and application thereof, and belongs to the field of medicinal chemistry. The sequence of the E6 targeting polypeptide is Glu-Leu-Thr-Leu-Gln-Glu-Leu-Leu-Gly-Glu-Glu-Arg. In the polypeptide, the first amino acid can be replaced by Tyr, the fourth and fifth amino acids can be Trp-Trp or Trp-Glu, the eighth amino acid is Tyr or Trp, and the twelfth amino acid is Glu. Compared with the prior art, the polypeptide can specifically target HPV16 E6 positive cells, has high selectivity, and can be prepared by a chemical synthesis method, has high purity, small molecular weight, high specificity, no immunogenicity and safety and reliability. The polypeptide can be used as a drug delivery carrier or coupled with a known drug component to form a drug combination, so that a better treatment effect can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to a polypeptide targeting HPV16 E6 protein and its preparation and application. BACKGROUND

[0002] High-grade squamous intraepithelial lesion (HSIL) is a cervical lesion associated with human papillomavirus (HPV) infection, 70% of which is caused by high-risk HPV (Hr-HPV). There are about 620,000 new cases of HSIL every year. Untreated HSIL will gradually evolve into cervical carcinoma in situ or even invasive cervical cancer after 5-10 years.

[0003] Persistent infection of Hr-HPV is a necessary factor for HSIL (high-grade squamous intraepithelial lesion) to develop into cervical cancer. At present, the most common treatment for HSIL is excisional treatment and ablation treatment of the cervix, but there is a risk of concurrent cervical stenosis, adhesion and dysfunction while eliminating abnormal tissue, and 5-25% of patients still have persistent infection of HPV after surgical treatment. In addition to surgical treatment, there is a lack of specific drugs for Hr-HPV in clinical practice. E6 cancer protein expressed by HPV16 plays an important role in the evolution of HSIL to cervical cancer and throughout the entire process of tumorigenesis.

[0004] Targeting polypeptides have small molecular weight, strong penetration blocking, and are easy to synthesize in large quantities. In addition, polypeptides can be repeatedly modified in structure to change their affinity, charge, hydrophilicity or hydrophobicity, stability and solubility. In tumor targeted therapy, targeting peptides show higher efficiency, safety, higher selectivity and are less likely to accumulate in the body. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a polypeptide targeting HPV16 E6 protein and its preparation and application.

[0006] The object of the present application can be achieved by the following technical solutions:

[0007] A polypeptide targeting HPV16 E6 protein, the amino acid sequence of which is:

[0008] SEQ.ID NO.1: Glu-Leu-Thr-Leu-Gln-Glu-Leu-Leu-Gly-Glu-Glu-Arg; or

[0009] The sequence of SEQ.ID NO.1 is adjusted as follows:

[0010] The first amino acid is replaced with Tyr;

[0011] The 4th and 5th amino acids can be Trp-Trp or Trp-Glu;

[0012] The 8th amino acid is Tyr or Trp;

[0013] The 12th amino acid is Glu.

[0014] Optionally, the amino acid sequence is:

[0015] SEQ. ID NO. 2: Tyr-Leu-Thr-Trp-Trp-Glu-Leu-Trp-Gly-Glu-Glu-Arg;

[0016] SEQ. ID NO. 3: Tyr-Leu-Thr-Trp-Gln-Glu-Leu-Leu-Gly-Glu-Glu-Glu; or

[0017] SEQ. ID NO. 4: Tyr-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gly-Glu-Glu-Glu.

[0018] The polypeptide described above is used in a drug delivery carrier for treating high-grade squamous intraepithelial lesions or preventing cervical cancer.

[0019] The polypeptide described above is used in a drug for treating high-grade squamous intraepithelial lesions or preventing cervical cancer.

[0020] A drug carrier comprising the polypeptide described above.

[0021] Optionally, the polypeptide is further coupled with a polypeptide sequence having self-assembly ability to form an environmentally corresponding polypeptide hydrogel.

[0022] A drug comprising the polypeptide described above and a drug component for treating high-grade squamous intraepithelial lesions or preventing cervical cancer coupled with the polypeptide.

[0023] Optionally, the drug component is:

[0024] Camptothecin, paclitaxel or cisplatin / carboplatin; or

[0025] PROTAC coupled with a linker and an E3 ubiquitin ligase ligand.

[0026] A preparation method of a polypeptide targeting HPV16 E6 protein, comprising the following steps:

[0027] By swelling the resin, the amino acids are sequentially coupled on the resin according to the amino acid sequence described above through the steps of deprotection and coupling;

[0028] Then, a lysis reagent is added to lyse the cells, and the polypeptide is obtained after centrifugation.

[0029] Optionally, the swelling comprises the following steps:

[0030] The Fmoc-Rink amide-MBHA Resin is weighed, swelled in DCM, and then filtered to remove the DCM, and then swelled in NMP for 30 minutes, and finally washed with NMP, DCM and NMP respectively.

[0031] Optionally, the deprotection comprises the following steps:

[0032] The swelled resin is placed in a reactor, and a piperidine / NMP solution containing HOBT is added for reaction, and then the solution is filtered after the reaction, and washed with NMP.

[0033] Optionally, the coupling comprises the following steps:

[0034] Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA are dissolved in NMP, and then the solution is added to the deprotected resin for reaction, and then the reaction solution is filtered after the reaction, and washed with DCM and NMP.

[0035] The beneficial effects of the present application are as follows:

[0036] The polypeptide of the present application can specifically target HPV16 E6 positive cells, has high selectivity, and can be prepared by chemical synthesis, has high purity, small molecular weight, strong specificity, no immunogenicity, and is safe and reliable. The polypeptide of the present application can be used as a drug delivery carrier, or coupled with known ingredients to form a drug combination, so as to obtain better therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0037] The present application will be further described below in conjunction with the accompanying drawings.

[0038] Figure 1 The affinity of the polypeptides of SEQ.ID NO.1-4 to the E6 protein was determined in Example 5 of the present application. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0040] Example 1

[0041] Solid phase synthesis of Glu-Leu-Thr-Leu-Gln-Glu-Leu-Leu-Gly-Glu-Glu-Arg (SEQ. ID NO. 1)

[0042] (1) Swelling of the resin

[0043] Weigh Fmoc-Rink amide-MBHA Resin 50mg (substitution 0.4mmol / g), swell in 7mL DCM for 30min, filter off the DCM, then swell in 10mL NMP for 30min, finally wash the resin with NMP, DCM, NMP 7mL respectively.

[0044] (2) Removal of Fmoc protecting group

[0045] Put the swelled resin into the reactor, add 7mL 0.1M HOBT in 25% piperidine / NMP (V / V) solution, react for 25min. After the reaction, filter off the solution, wash the resin with NMP. The resin with the initial attached Fmoc protecting group removed is obtained.

[0046] (3) Synthesis of Fmoc-Arg(Pbf)-Rink amide-MBHA Resin

[0047] Dissolve Fmoc-Arg(Pbf)-OH (0.04mmol), HBTU (0.04mmol), HOBT (0.04mmol) and DIPEA (0.08mmol) in 10mL NMP, then add the solution into the resin above, react for 45min. After the reaction, filter off the solution, wash the resin with DCM and NMP 7mL respectively for 3 times.

[0048] (4) Detection of coupling efficiency

[0049] Detect the coupling efficiency of the resin by ninhydrin method or bromophenol blue method, if the color reaction is negative, then enter the next coupling cycle.

[0050] Ninhydrin method: take a small amount of resin particles, wash with ethanol, put into a transparent vial, add 5% ninhydrin ethanol, 2 drops of 80% phenol ethanol solution, 2 drops of KCN pyridine solution (2ml 0.001M KCN diluted in 98ml pyridine), heat at 100℃ for 5min, if the resin shows blue color, it is positive.

[0051] Bromophenol blue method: take a small amount of resin particles, wash with dimethylglycolamide, put into a transparent vial, add 3 drops of 1% bromophenol blue dimethylglycolamide solution, shake at room temperature for 3min, if the resin shows blue color, it is positive.

[0052] (5) Extension of the peptide chain

[0053] According to the sequence of the polypeptide, repeat the above-mentioned deprotection and coupling steps to sequentially connect the corresponding amino acids to obtain the resin connected with the polypeptide sequence of (SEQ. ID NO. 1).

[0054] (6) Cleavage of the polypeptide on the resin

[0055] The resin connected with the polypeptide sequence of (SEQ. ID NO. 1) obtained above was placed in a reaction bottle, and 10 mL of cleavage reagent Reagent K (TFA / benzyl mercaptan / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added, and then shaken at 0°C for 30 min, and then reacted at room temperature for 3 h. After the reaction was completed, the filtrate was filtered, washed with a small amount of TFA and DCM three times, and then combined. The filtrate was added to a large amount of ice ethyl ether to precipitate a white flocculent precipitate, and the target polypeptide was obtained by freeze centrifugation.

[0056] The polypeptide crude product obtained above was dissolved in 2 mL of water, and directly subjected to preparative liquid chromatography purification, and the chromatography conditions were as follows: C18 reverse phase column (320 mm x 28 mm, 5 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 20% to 80%, 20 min; flow rate 6 mL / min, detection wavelength 214 nm. The collected solution was freeze-dried to obtain 25 mg of pure product. The theoretical relative molecular mass was 1429.564. ESI-MS m / z: found [M+3H] 3+ = 1432.52, 1 / 2 [M+2H] 2+ = 715.57.

[0057] Example 2

[0058] Solid phase synthesis of Tyr-Leu-Thr-Trp-Trp-Glu-Leu-Trp-Gly-Glu-Glu-Arg (SEQ. ID NO. 2)

[0059] The theoretical relative molecular mass was 1667.824. ESI-MS m / z: found [M+2H] 2+ = 1669.71, 1 / 2 [M+2H] 2+ = 834.78.

[0060] Example 3

[0061] Solid phase synthesis of Tyr-Leu-Thr-Trp-Gln-Glu-Leu-Leu-Gly-Glu-Glu-Glu (SEQ. ID NO. 3)

[0062] Theoretical relative molecular mass is 1510.594. ESI-MS m / z: found [M+2H] 2+ = 1512.15.

[0063] Example 4

[0064] Solid phase synthesis of Tyr-Leu-Thr-Trp-Gln-Glu-Leu-Tyr-Gly-Glu-Glu-Glu (SEQ. ID NO. 4)

[0065] Theoretical relative molecular mass is 1617.714. ESI-MS m / z: found [M+2H] 2+ = 1619.23, 1 / 2 [M+Na+H] 2+ = 820.23.

[0066] Example 5

[0067] Surface plasmon resonance (SPR) method was used to detect the binding of SEQ. ID NO. 1, SEQ. ID NO. 2, SEQ. ID NO. 3, SEQ. ID NO. 4 to E6 protein, respectively.

[0068] Human recombinant protein E6 was coupled to a CM 5 chip, and the analyte was diluted to a concentration gradient (500, 250, 125, 62.5, 31.25 nM). Appropriate regeneration conditions (such as Glycine 2.5) were used, the flow rate was set to 30 μL / min, the injection time for each cycle was set to 90 s, and the dissociation time was also 90 s. According to the Biacore instrument procedure, multi-cycle kinetic test was carried out, and finally the binding and dissociation curves were analyzed by Biacore T200 Evaluation Software to calculate the affinity related parameters.

[0069] The results are shown in Table 1. Figure 1 The equilibrium dissociation constant K D of SEQ. ID NO. 1 peptide to E6 is 23.16 nM, the equilibrium dissociation constant K D of SEQ. ID NO. 2 peptide to E6 is 4.836 nM, the equilibrium dissociation constant K D of SEQ. ID NO. 3 peptide to E6 is 85.53 nM, and the equilibrium dissociation constant K D of SEQ. ID NO. 4 peptide to E6 is 89.13 nM, indicating that the peptide sequence of the present application has strong binding to E6 and can be used as a polypeptide targeting E6.

[0070] As described above, the polypeptide of the present application can effectively target HPV16 E6 positive cells, and thus has the potential as a pharmaceutical ingredient or a pharmaceutical carrier for the prevention or treatment of HPV infection.

[0071] Specifically, in some examples of the present application, the polypeptide described above and the polypeptide sequence having self-assembly ability can be coupled to form a corresponding polypeptide hydrogel, thereby forming a drug delivery system capable of targeting HPV16 E6 positive cells. More specifically, the hydrogel described above can be wrapped with some known chemotherapeutic drugs, and after wrapping, the drugs can be accurately delivered to HPV16 E6 positive cells through the targeting effect of the polypeptide sequence of the present application.

[0072] In other examples of the present application, the polypeptide described above can also be coupled with known effective anticancer drugs, so that the formed drug combination has the function of targeting HPV16 E6 positive cells and improves the anticancer effect of the drug ingredient. More specifically, the anticancer drug can be a chemotherapeutic drug, such as, but not limited to, camptothecin, paclitaxel, or cisplatin / carboplatin, etc.; or a PROTAC coupled with a linker and an E3 ubiquitin ligase ligand.

[0073] In the description of the present specification, the description of the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0074] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only illustrative of the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1.A polypeptide targeting HPV16 E6 protein, with an amino acid sequence of: SEQ. ID NO. 2: Tyr-Leu-Thr-Trp-Trp-Glu-Leu-Trp-Gly-Glu-Glu-Arg. 2.A method for preparing a polypeptide targeting HPV16 E6 protein, comprising the following steps: connecting amino acids in sequence according to the amino acid sequence of SEQ. ID NO. 2 on a resin by swelling treatment of the resin, deprotection and coupling; and adding a cleavage agent for cleavage, and obtaining the polypeptide after centrifugation. The swelling comprises the following steps: weighing Fmoc-Rink amide-MBHA Resin, swelling the resin in DCM, filtering the DCM, swelling the resin in NMP for 30 min, and then washing the resin with NMP, DCM and NMP respectively. The deprotection comprises the following steps: placing the swelled resin in a reactor, adding a piperidine / NMP solution containing HOBT for reaction, filtering the solution after the reaction, and washing the resin with NMP. The coupling comprises the following steps: dissolving Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA in NMP, adding the solution to the resin after deprotection for reaction, filtering the reaction solution after the reaction, and washing the resin with DCM and NMP. ​ 3. The production method according to claim 2, characterized by, ​ ​ ​ ​ ​ ​

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